US20090118383A1 - Production method of porous resin bead - Google Patents
Production method of porous resin bead Download PDFInfo
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- US20090118383A1 US20090118383A1 US12/264,940 US26494008A US2009118383A1 US 20090118383 A1 US20090118383 A1 US 20090118383A1 US 26494008 A US26494008 A US 26494008A US 2009118383 A1 US2009118383 A1 US 2009118383A1
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- porous resin
- resin bead
- acrylate
- acyloxystyrene
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- GFAYATKJQOQVFZ-IVZWLZJFSA-N CC[C@H]1O[C@@H](C)C[C@H]1OC(=O)CCC(=O)OC Chemical compound CC[C@H]1O[C@@H](C)C[C@H]1OC(=O)CCC(=O)OC GFAYATKJQOQVFZ-IVZWLZJFSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F6/00—Post-polymerisation treatments
- C08F6/001—Removal of residual monomers by physical means
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F2/00—Processes of polymerisation
- C08F2/12—Polymerisation in non-solvents
- C08F2/16—Aqueous medium
- C08F2/18—Suspension polymerisation
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/10—Esters
- C08F220/20—Esters of polyhydric alcohols or phenols, e.g. 2-hydroxyethyl (meth)acrylate or glycerol mono-(meth)acrylate
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F8/00—Chemical modification by after-treatment
- C08F8/12—Hydrolysis
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F8/00—Chemical modification by after-treatment
- C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
- C08J9/16—Making expandable particles
- C08J9/20—Making expandable particles by suspension polymerisation in the presence of the blowing agent
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F212/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring
- C08F212/02—Monomers containing only one unsaturated aliphatic radical
- C08F212/04—Monomers containing only one unsaturated aliphatic radical containing one ring
- C08F212/06—Hydrocarbons
- C08F212/08—Styrene
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F2810/00—Chemical modification of a polymer
- C08F2810/50—Chemical modification of a polymer wherein the polymer is a copolymer and the modification is taking place only on one or more of the monomers present in minority
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2203/00—Foams characterized by the expanding agent
- C08J2203/12—Organic compounds only containing carbon, hydrogen and oxygen atoms, e.g. ketone or alcohol
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2203/00—Foams characterized by the expanding agent
- C08J2203/14—Saturated hydrocarbons, e.g. butane; Unspecified hydrocarbons
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2325/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring; Derivatives of such polymers
- C08J2325/02—Homopolymers or copolymers of hydrocarbons
- C08J2325/04—Homopolymers or copolymers of styrene
- C08J2325/08—Copolymers of styrene
Definitions
- the present invention relates to a production method of a porous resin bead that can be suitably used, for example, as a support for solid phase synthesis.
- a polystyrene-based porous resin bead there are a hydroxystyrene-polyene copolymer (see, Patent Documents 1 and 2) and a copolymer resin obtained by copolymerizing an alkoxystyrene, an aromatic polyvinyl and an aromatic vinyl compound (see, Patent Document 3).
- These porous resin beads are mainly used as an ion exchange resin, an adsorbent or the like.
- porous resin beads have been recently used also as a support for solid phase synthesis, which is a reaction site allowing efficient progress of a chemical synthesis reaction.
- the positions at which the chemical synthesis reaction starts are considered to be functional groups distributed in a crosslinked network structure of the porous resin bead. Accordingly, there is strongly needed a porous resin having an appropriately-sized crosslinked network structure and an appropriate distribution of functional groups according to the kind or amount of a substance to be chemically synthesized.
- Patent Document 1 JP-A-52-023193
- Patent Document 2 JP-A-58-210914
- Patent Document 3 JP-A-05-086132
- Patent Document 4 JP-A-2005-097545
- the present invention has been made under these circumstances and an object of the present invention is to provide a method for producing a porous resin bead which can be suitably used as a support for solid phase synthesis that allows efficient progress of a chemical synthesis reaction, and particularly, as a solid phase support enabling solid phase synthesis of oligonucleotide with high purity at a high synthesis rate.
- a method for producing a porous resin bead containing an aromatic vinyl compound-hydroxystyrene-di(meth)acrylate copolymer including: dissolving a monomer mixture containing an aromatic vinyl compound, an acyloxystyrene and a di(meth)acrylate compound, and a polymerization initiator in an organic solvent to obtain a solution containing the monomer mixture and the polymerization initiator; suspending the solution in water in the presence of a dispersion stabilizer; performing a suspension copolymerization to thereby obtain an aromatic vinyl compound-acyloxystyrene-di(meth)acrylate compound copolymer; and hydrolyzing an acyloxy group of the acyloxystyrene monomer component in the copolymer.
- the di(meth)acrylate compound is preferably at least one member selected from the group consisting of a (poly)ethylene glycol di(meth)acrylate and an alkanediol di(meth)acrylate.
- the monomer mixture preferably contains the acyloxystyrene in a proportion of 80 to 800 ⁇ mol/g and the di(meth)acrylate compound in a proportion of 200 to 2,000 ⁇ mol/g.
- the organic solvent is at least one member selected from the group consisting of a hydrocarbon and an alcohol, and the organic solvent is used in a weight ratio of 0.5 to 2.5 based on the monomer mixture.
- an aromatic vinyl compound-acyloxystyrene-di(meth)acrylate compound copolymer is produced by a suspension copolymerization of a monomer mixture containing an aromatic vinyl compound, an acyloxystyrene and a di(meth)acrylate compound in predetermined proportions, respectively, and the acyloxy group of the acyloxystyrene monomer component in the copolymer is then hydrolyzed, whereby a porous resin bead containing an aromatic vinyl compound-hydroxystyrene-di(meth)acrylate copolymer is obtained.
- the porous resin bead obtained by the method of the present invention has appropriate crosslink density and distribution of hydroxyl groups. Therefore, when this bead is used as a support for solid phase synthesis of oligonucleotide, the reactions performed on the support are not inhibited with each other, so that oligonucleotide can be produced through a solid-phase synthesis with high purity at a high synthesis rate.
- oligonucleotide can be obtained in a high synthesis yield while keeping the proportion of full-length oligonucleotide high.
- the method for producing a porous resin bead containing an aromatic vinyl compound-hydroxystyrene-di(meth)acrylate copolymer of the present invention includes dissolving a monomer mixture containing an aromatic vinyl compound, an acyloxystyrene and a di(meth)acrylate compound, and a polymerization initiator in an organic solvent to obtain a solution of the monomer mixture and the polymerization initiator; suspending the solution in water in the presence of a dispersion stabilizer; performing a suspension copolymerization to thereby obtain an aromatic vinyl compound-acyloxystyrene-di(meth)acrylate compound copolymer; and hydrolyzing the acyloxy group of the acyloxystyrene monomer component in the obtained copolymer.
- examples of the aromatic vinyl compound include, but are not limited to, styrene, a nucleus alkyl-substituted styrene such as o-methylstyrene, m-methylstyrene, p-methylstyrene, 2,4-dimethylstyrene, ethylstyrene and p-tert-butylstyrene, an ⁇ -alkyl-substituted styrene such as ⁇ -methylstyrene and ⁇ -methyl-p-methylstyrene, and a nucleus halogenated styrene such as chlorostyrene.
- styrene is preferably used as the aromatic vinyl compound.
- the acyloxystyrene may have, in addition to the acyloxy group, one or a plurality of arbitrary substituents on the aromatic nucleus so long as they do not adversely affect the suspension copolymerization or give no harmful effect when the obtained porous bead is used as a support for solid phase synthesis.
- substituents include an alkyl group or an alkoxyl group having a carbon number of 1 to 5, a halogen atom and a nitro group.
- the position of the acyloxy group on the aromatic nucleus of the acyloxystyrene with respect to the vinyl group is not particularly limited but is preferably the para-position. That is, in the present invention, the acyloxystyrene is preferably a p-acyloxystyrene. Further, the alkyl group in the acyl group preferably has a carbon number of 1 to 5. Particularly, in the present invention, p-acetoxystyrene is preferably used as the acyloxystyrene.
- the di(meth)acrylate compound means a dimethacrylate or a diacrylate and is preferably at least one member selected from the group consisting of a (poly)ethylene glycol di(meth)acrylate and an alkanediol di(meth)acrylate.
- the (poly)ethylene glycol di(meth)acrylate include ethylene glycol di(meth)acrylate, diethylene glycol di(meth)acrylate, triethylene glycol di(meth)acrylate, tetraethylene glycol di(meth)acrylate, polyethylene glycol di(meth)acrylate, propylene glycol di(meth)acrylate, dipropylene glycol di(meth)acrylate, tripropylene glycol di(meth)acrylate, and polypropylene glycol di(meth)acrylate.
- the alkane in the alkanediol di(meth)acrylate preferably has a carbon number of 4 to 10.
- specific examples of the alkanediol di(meth)acrylate include 1,2-butanediol di(meth)acrylate, 1,3-butanediol di(meth)acrylate, 1,4-butanediol di(meth)acrylate, 1,5-pentanediol di(meth)acrylate, 1,6-hexanediol di(meth)acrylate, 1,9-nonanediol di(meth)acrylate, 1,10-decanediol di(meth)acrylate, 2-methyl-1,8-octanediol di(meth)acrylate, and 1,4-cyclohexanediol di(meth)acrylate.
- the acyloxystyrene in the monomer mixture of an aromatic vinyl compound, an acyloxystyrene and a di(meth)acrylate compound, which is used for the suspension copolymerization, is preferably contained in a proportion of 80 to 800 ⁇ mol/g, and more preferably from 200 to 500 ⁇ mol/g. If the proportion of the acyloxystyrene in the monomer mixture is less than 80 ⁇ mol/g, the porous resin bead to be obtained is low in the amount of the hydroxyl group and when used as a support for solid phase synthesis, the amount of the synthesis reaction product to be obtained is small.
- the proportion of the di(meth)acrylate compound is preferably from 200 to 2,000 ⁇ mol/g, and more preferably from 300 to 1,500 ⁇ mol/g. If the proportion of the di(meth)acrylate compound in the monomer mixture is less than 200 ⁇ mol/g, the specific surface area of the porous resin bead to be obtained is extremely small and when used as a support for solid phase synthesis, the amount of the synthesis reaction product to be obtained is small. On the other hand, if the proportion of the di(meth)acrylate compound in the monomer mixture exceeds 2,000 ⁇ mol/g, since the swelling degree of the porous resin bead to be obtained in an organic solvent is low, the amount of the synthesis reaction product to be obtained is small.
- the suspension copolymerization of the monomer mixture containing an aromatic vinyl compound, an acyloxystyrene and a di(meth)acrylate compound can be performed by a conventionally known normal method.
- the monomer mixture and a polymerization initiator are dissolved in an organic solvent, a dispersion stabilizer is separately dissolved in water, these two solutions are mixed in a nitrogen stream, the mixed solution is stirred so that the solution obtained by dissolving the monomer mixture and the polymerization initiator in the organic solvent can be dispersed as fine droplets in water, and the copolymerization is performed with stirring by raising the temperature.
- the suspension copolymerization reaction may be performed usually with stirring at a reaction temperature of 60 to 90° C. over 0.5 to 48 hours, but the copolymerization reaction conditions are not limited thereto.
- the hydrocarbon includes an aliphatic hydrocarbon and an aromatic hydrocarbon, and the aliphatic hydrocarbon may be either saturated or unsaturated but is preferably an aliphatic saturated hydrocarbon having a carbon number of 5 to 12 or an alkylbenzene.
- the aliphatic saturated hydrocarbon having a carbon number of 5 to 12 include n-hexane, n-heptane, n-octane, isooctane, undecane and dodecane.
- the alkylbenzene include toluene.
- the alcohol is preferably an aliphatic alcohol, and more preferably an aliphatic alcohol having a carbon number of 5 to 12.
- Specific preferred examples thereof include 2-ethylhexyl alcohol, tert-amyl alcohol, nonyl alcohol, 2-octanol, decanol, lauryl alcohol and cyclohexanol.
- one of these hydrocarbons and alcohols may be used alone, or the hydrocarbon and the alcohol may be used in combination.
- the organic solvent is usually used in a weight ratio of organic solvent/monomer of 0.5 to 2.5, preferably from 1.0 to 2.0. If the weight ratio of organic solvent/monomer is less than or exceeds the above-mentioned range, in either case, the specific surface area of the porous resin bead to be obtained becomes small and, for example, when used as a support for solid phase synthesis, the amount of the synthesis reaction product by a chemical reaction is reduced.
- the dispersion stabilizer is not particularly limited, and an arbitrary dispersion stabilizer may be used so long as it is conventionally used as a dispersion stabilizer in the suspension polymerization.
- the dispersion stabilizer include a hydrophilic protective colloid agent such as polyvinyl alcohol, polyacrylic acid, gelatin, starch and carboxyl methyl cellulose; and a hardly-soluble inorganic powder such as calcium carbonate, magnesium carbonate, calcium phosphate, barium sulfate, calcium sulfate and bentonite.
- the amount of the dispersion stabilizer used is preferably from 0.01 to 10 wt % based on the water used in the suspension polymerization.
- the amount of the dispersion stabilizer is less than 0.01 wt % based on the water used in the suspension polymerization, the stability of suspension polymerization is impaired and a large amount of an aggregate is produced. On the other hand, if the amount of the dispersion stabilizer exceeds 10 wt % based on the water used in the suspension polymerization, a large number of particles which are too fine to use as a support for solid phase synthesis are produced.
- the polymerization initiator for use in the suspension polymerization is not particularly limited, and an arbitrary polymerization initiator may be used so long as it is conventionally used as a polymerization initiator in suspension polymerization.
- the polymerization initiator include a peroxide such as dibenzoyl peroxide, dilauroyl peroxide, distearoyl peroxide, 1,1-di(tert-butylperoxy)-2-methylcyclohexane, 1,1-di(tert-hexylperoxy)-3,3,5-trimethylcyclohexane, 1,1-di(tert-hexylperoxy)cyclohexane, 1,1-di(tert-butylperoxy)cyclohexane, di-tert-hexyl peroxide, tert-butylcumyl peroxide, di-tert-butyl peroxide, 1,1,3,3-tetramethylbutylperoxy-2-e
- the organic solvent in which the monomer mixture and the polymerization initiator are dissolved is suspended in water and subjected to suspension copolymerization to precipitate a copolymer. Thereafter, the copolymer is purified by washing to remove monomers remaining in the copolymer and impurities such as organic solvent, dispersion stabilizer and polymerization initiator.
- the solvent for washing is not particularly limited so long as it is a solvent useful for the removal of those impurities, but water, methanol, ethanol, acetonitrile, acetone, toluene, hexane, tetrahydrofuran or the like is usually used.
- this may be attained, for example, by suction-filtering the liquid dispersion of the copolymer after suspension copolymerization, washing the resulting copolymer with stirring in the solvent for washing, and repeating the operation of suction-filtering and subsequent washing of the copolymer in a similar manner.
- the system may be heated, if necessary, to remove volatile impurities in the copolymer.
- an aromatic vinyl compound-acyloxystyrene-di(meth)acrylate compound copolymer is firstly obtained in this way, and the acyloxy group of the acyloxystyrene monomer component in the copolymer is then hydrolyzed, whereby the objective porous resin bead containing an aromatic vinyl compound-hydroxystyrene-di(meth)acrylate copolymer can be obtained.
- acyloxy group of the acyloxystyrene monomer component in the aromatic vinyl compound-acyloxystyrene-di(meth)acrylate compound copolymer conventionally known methods and conditions may be employed. However, in the present invention, it is not necessary to hydrolyze the acyloxy groups of all acyloxystyrene monomer components in the obtained aromatic vinyl compound-acyloxystyrene-di(meth)acrylate compound copolymer, and it may be usually sufficient if a part of the acyloxy groups are hydrolyzed.
- the catalyst used for the hydrolysis of the acyloxy group of the acyloxystyrene monomer component in the aromatic vinyl compound-acyloxystyrene-di(meth)acrylate compound copolymer may be an acid catalyst such as hydrochloric acid and hydrobromic acid, or an alkali catalyst such as hydrazine, sodium hydroxide, tetramethylammonium hydroxide and aqueous ammonia.
- Preferred examples of the reaction solvent used for the hydrolysis include, but are not limited to, tetrahydrofuran, ethanol, methanol, dioxane and water.
- the reaction conditions of the hydrolysis may be appropriately set.
- the amount of sodium hydroxide used is from 1 to 10 equivalents of the acyloxystyrene monomer component in the aromatic vinyl compound-acyloxystyrene-di(meth)acrylate compound copolymer
- the amount of ethanol used is from 1 to 10 times the weight of water used, and the reaction is performed at 50 to 80° C. for 1 to 12 hours.
- the amount of sodium hydroxide used is from 1 to 5 equivalents of the acyloxystyrene monomer component, and the reaction is performed at 50 to 80° C. for 1 to 6 hours.
- the ester derived from the di(meth)acrylate compound is hydrolyzed and the solvent resistance of the porous resin bead is impaired.
- the reaction mixture after hydrolysis may be adjusted to near neutrality by using an acid or an alkali and then washed by the same method as described above to remove impurities.
- an aromatic vinyl compound-acyloxystyrene-di(meth)acrylate compound copolymer can be obtained through a suspension copolymerization of a monomer mixture containing an aromatic vinyl compound, an acyloxystyrene and a di(meth)acrylate compound, and a porous resin bead containing an aromatic vinyl compound-hydroxystyrene-di(meth)acrylate compound copolymer can be produced by hydrolyzing the acyloxy group of the acyloxystyrene monomer component in the obtained copolymer. Thereafter, if desired, the porous resin bead may be further subjected to a treatment such as drying or classification.
- the thus-obtained porous resin bead can be suitably used as a support for solid phase synthesis to synthesize, for example, oligonucleotide.
- the oligonucleotide synthesis using the porous resin bead according to the present invention as a support for solid phase synthesis can be performed by a conventionally known method.
- a linker is bound to the hydroxy group of the porous resin bead of the present invention, and amidite units are then bound one by one to give a predetermined base sequence from the end of the linker.
- This synthesis reaction can be performed using an automatic synthesis apparatus.
- a linker-bound porous resin particle is packed in a flow-type reactor of the apparatus, various organic solvents such as acetonitrile and an amidite solution are sequentially fed thereto, and the reaction is repeated. Finally, the linker portion is cut off by hydrolysis or the like, whereby the objective oligonucleotide can be obtained.
- a conventionally known linker is appropriately used.
- the oligonucleotide can be synthesized by binding a nucleoside linker having a structure shown below to the porous resin particle of the present invention.
- the circle represents the porous resin bead containing an aromatic vinyl compound-hydroxystyrene-di(meth)acrylate copolymer, which is obtained by the method of the present invention
- DMT represents a protective dimethoxytrityl group at the 5′-position
- B 1 represents a base.
- a 500 mL-volume separable flask equipped with a condenser, a stirrer and a nitrogen inlet tube was placed in a constant temperature water bath, and 2.5 g of polyvinyl alcohol and 250 g of distilled water were charged thereto and dissolved with stirring at 300 rpm to prepare an aqueous polyvinyl alcohol solution.
- This solution was added to the aqueous polyvinyl alcohol solution prepared above and after stirring at a stirring rate of 500 rpm in a nitrogen stream at room temperature, suspended in water, and suspension copolymerization was performed for 8 hours by raising the temperature to 80° C.
- the obtained copolymer was washed by filtration using distilled water and acetone and then dispersed in acetone to make a total amount of about 1 L.
- the resulting liquid dispersion was then allowed to stand still until the bead-like copolymer was precipitated so that the precipitate was not disturbed even when the liquid was tilted, and the supernatant acetone was then discarded.
- the operation of again adding acetone to the precipitate to make a total amount of about 500 mL, allowing the liquid dispersion to stand still, and then discarding acetone was repeated 10 times or more, thereby performing the classification.
- the thus-obtained liquid dispersion was filtered, and the residue was dried under reduced pressure to obtain a porous resin bead composed of a styrene-p-acetoxystyrene-ethylene glycol dimethacrylate copolymer, as powder.
- a 500 mL-volume separable flask equipped with a condenser, a stirrer and a nitrogen inlet tube was dipped in a constant temperature water bath, and 20 g of the porous resin bead composed of a styrene-p-acetoxystyrene-ethylene glycol dimethacrylate copolymer, obtained above as powder, and 100 g of ethanol were charged thereto and dispersed with stirring. Subsequently, an aqueous solution prepared by dissolving 1 g of sodium hydroxide in 50 g of distilled water was added, and hydrolysis reaction was then performed for 3 hours by raising the temperature to 75° C.
- the obtained reaction mixture was neutralized with hydrochloric acid and washed by filtration using distilled water and acetone. Finally, the obtained liquid dispersion was filtered and further dried under reduced pressure to obtain a porous resin bead composed of a styrene-p-hydroxystyrene-ethylene glycol dimethacrylate copolymer, as powder.
- the obtained porous resin bead was subjected to the following analyses.
- the median diameter was measured by a laser diffraction/scattering method (a laser diffraction/scattering particle size distribution analyzer, LA-920, manufactured by Horiba Ltd.).
- the average pore diameter was measured by a mercury penetration method (a mercury porosimeter, PoreMaster 60GT, manufactured by Quantachrome Instruments).
- the amount of hydroxy group was measured by titration according to JIS K 0070. That is, the hydroxy group of the porous resin bead to be measured was acetylated with a known amount of an acetylation reagent (acetic anhydride/pyridine), and the amount of the acetic anhydride not consumed in the actylation was determined by titration using potassium hydroxide, whereby the amount of the hydroxy group in the sample was calculated. More specifically, this was performed as follows.
- the acetylation reagent was prepared by adding pyridine to 25 g of acetic anhydride to make a total amount of 100 mL, from 0.5 to 2 g of the measurement sample (dried porous resin bead) was weighed in a flask, and 0.5 mL of the acetylation reagent and 4.5 mL of pyridine were exactly added to the flask. The mixture in the flask was kept at 95 to 100° C. for 2 hours and then allowed to cool to room temperature, and 1 mL of distilled water was added thereto. After heating for 10 minutes, the acetic anhydride not consumed in the acetylation was decomposed. The entire content in the flask was transferred to a beaker, diluted with distilled water to a total amount of 150 mL and then titrated with an aqueous 0.5 N sodium hydroxide solution.
- a blank was measured by the same operation as above without charging the measurement sample.
- the amount of the hydroxy group in the measurement sample was calculated according to the following formula (I), wherein A ( ⁇ mol/g) is the amount of the hydroxyl group in the measurement sample, B (mL) is the titer of the aqueous potassium hydroxide solution when measuring a blank, C (mL) is the titer of the aqueous potassium hydroxide solution when measuring the measurement sample, f is the factor of the aqueous potassium hydroxide solution, and M (g) is the weight of the measurement sample weighed.
- the median diameter of the porous resin bead was 80 ⁇ m, the average pore diameter was 29 nm, and the amount of the hydroxy group was 491 ⁇ mol/g.
- a porous resin bead composed of a styrene-p-hydroxystyrene-triethylene glycol dimethacrylate copolymer was obtained as powder in the same manner as in Example 1 except that in Example 1, 40 g of styrene, 3.5 g (proportion in the monomer mixture: 369 ⁇ mol/g) of p-acetoxystyrene, 15 g (proportion in the monomer mixture: 897 ⁇ mol/g) of triethylene glycol dimethacrylate, 80 g of 1-undecanol and 1 g of benzoyl peroxide (25% aqueous product) were used.
- the obtained porous resin bead was analyzed in the same manner as in Example 1, and as a result, the median diameter was 75 ⁇ m, the average pore diameter was 34 nm, and the amount of the hydroxy group was 393 ⁇ mol/g.
- porous resin bead (1 g) obtained above, 0.18 g of DMT-dT-3′-succinate (produced by Beijing OM Chemicals), 0.09 g of HBTU (produced by Novabiochem), 0.084 mL of N,N-diisopropylethylamine and 10 mL of acetonitrile were mixed and reacted with stirring at room temperature for 12 hours, and the reaction mixture was washed by filtering using acetonitrile and dried.
- the addition amount of the DMT-dT-3′-succinate determined by measuring the absorbance (412 nm) of the DMT group deprotected using a p-toluenesulfonic acid/acetonitrile solution is shown in Table 1.
- the DMT-dT-3′-succinate-bound porous resin bead obtained above was packed in a column, the column was set in an Applied Biosystems 3400 DNA synthesizer (manufactured by Applied Biosystems), and synthesis of oligonucleotide dT20 was performed under the conditions of a synthesis scale of 1 ⁇ mol and DMT-off.
- the column after the synthesis was dried, and the weight yield (mg/ ⁇ mol) of the obtained oligonucleotide determined by the weight measurement is shown in Table 1.
- a porous resin bead composed of a styrene-p-hydroxystyrene-1,6-hexanediol dimethacrylate copolymer was obtained as powder in the same manner as in Example 1 except that in Example 1, 40 g of styrene, 3.5 g (proportion in the monomer mixture: 369 ⁇ mol/g) of p-acetoxystyrene, 15 g (proportion in the monomer mixture: 1,008 ⁇ mol/g) of 1,6-hexanediol dimethacrylate, 75 g of 1-decanol and 1 g of benzoyl peroxide (25% aqueous product) were used.
- the obtained porous resin bead was analyzed in the same manner as in Example 1, and as a result, the median diameter was 72 ⁇ m, the average pore diameter was 26 nm, and the amount of the hydroxy group was 446 ⁇ mol/g.
- a porous resin bead composed of a styrene-p-hydroxystyrene-1,9-nonanediol diacrylate copolymer was obtained as powder in the same manner as in Example 1 except that in Example 1, 48 g of styrene, 2.5 g (proportion in the monomer mixture: 264 ⁇ mol/g) of p-acetoxystyrene, 8 g (proportion in the monomer mixture: 510 ⁇ mol/g) of 1,9-nonanediol diacrylate, 90 g of 1-nonanol and 1 g of benzoyl peroxide (25% aqueous product) were used.
- a porous resin bead composed of a styrene-p-hydroxystyrene-divinylbenzene copolymer was obtained as powder in the same manner as in Example 1 except that in Example 1, 48 g of styrene, 3.5 g (proportion in the monomer mixture: 369 ⁇ mol/g) of p-acetoxystyrene, 7 g (proportion in the monomer mixture: 506 ⁇ mol/g) of divinylbenzene (55%), 52 g of 2-ethylhexanol, 23 g of isooctane and 1 g of benzoyl peroxide (25% aqueous product) were used.
- the obtained porous resin bead was analyzed in the same manner as in Example 1, and as a result, the median diameter was 86 ⁇ m, the average pore diameter was 33 nm, and the amount of the hydroxy group was 422 ⁇ mol/g.
- a porous resin bead composed of a styrene-p-hydroxystyrene-divinylbenzene copolymer was obtained as powder in the same manner as in Example 1 except that in Example 1, 48 g of styrene, 3.5 g (proportion in the monomer mixture: 369 ⁇ mol/g) of p-acetoxystyrene, 7 g (proportion in the monomer mixture: 506 ⁇ mol/g) of divinylbenzene (55%), 75 g of 1-decanol and 1 g of benzoyl peroxide (25% aqueous product) were used.
- the obtained porous resin bead was analyzed in the same manner as in Example 1, and as a result, the median diameter was 116 ⁇ m, the average pore diameter was 68 nm, and the amount of the hydroxy group was 432 ⁇ mol/g.
Abstract
The present invention provides a method for producing a porous resin bead containing an aromatic vinyl compound-hydroxystyrene-di(meth)acrylate copolymer, the method including: dissolving a monomer mixture containing an aromatic vinyl compound, an acyloxystyrene and a di(meth)acrylate compound, and a polymerization initiator in an organic solvent to obtain a solution containing the monomer mixture and the polymerization initiator; suspending the solution in water in the presence of a dispersion stabilizer; performing a suspension copolymerization to thereby obtain an aromatic vinyl compound-acyloxystyrene-di(meth)acrylate compound copolymer; and hydrolyzing an acyloxy group of the acyloxystyrene monomer component in the copolymer. The porous resin bead produced by the method of the present invention can be suitably used, for example, as a support for solid phase synthesis.
Description
- The present invention relates to a production method of a porous resin bead that can be suitably used, for example, as a support for solid phase synthesis.
- Conventionally, as a polystyrene-based porous resin bead, there are a hydroxystyrene-polyene copolymer (see, Patent Documents 1 and 2) and a copolymer resin obtained by copolymerizing an alkoxystyrene, an aromatic polyvinyl and an aromatic vinyl compound (see, Patent Document 3). These porous resin beads are mainly used as an ion exchange resin, an adsorbent or the like. Accordingly, in such applications, it is heretofore an important target to enhance the substance adsorbing ability per unit volume of the porous resin bead by imparting as much a functional group as possible to the porous resin bead or increasing the specific surface area of the porous resin bead as large as possible.
- On the other hand, such porous resin beads have been recently used also as a support for solid phase synthesis, which is a reaction site allowing efficient progress of a chemical synthesis reaction. In such a case, the positions at which the chemical synthesis reaction starts are considered to be functional groups distributed in a crosslinked network structure of the porous resin bead. Accordingly, there is strongly needed a porous resin having an appropriately-sized crosslinked network structure and an appropriate distribution of functional groups according to the kind or amount of a substance to be chemically synthesized.
- Patent Document 1: JP-A-52-023193
- Patent Document 2: JP-A-58-210914
- Patent Document 3: JP-A-05-086132
- Patent Document 4: JP-A-2005-097545
- The present invention has been made under these circumstances and an object of the present invention is to provide a method for producing a porous resin bead which can be suitably used as a support for solid phase synthesis that allows efficient progress of a chemical synthesis reaction, and particularly, as a solid phase support enabling solid phase synthesis of oligonucleotide with high purity at a high synthesis rate.
- According to the present invention, there is provides a method for producing a porous resin bead containing an aromatic vinyl compound-hydroxystyrene-di(meth)acrylate copolymer, the method including: dissolving a monomer mixture containing an aromatic vinyl compound, an acyloxystyrene and a di(meth)acrylate compound, and a polymerization initiator in an organic solvent to obtain a solution containing the monomer mixture and the polymerization initiator; suspending the solution in water in the presence of a dispersion stabilizer; performing a suspension copolymerization to thereby obtain an aromatic vinyl compound-acyloxystyrene-di(meth)acrylate compound copolymer; and hydrolyzing an acyloxy group of the acyloxystyrene monomer component in the copolymer.
- In the present invention, the di(meth)acrylate compound is preferably at least one member selected from the group consisting of a (poly)ethylene glycol di(meth)acrylate and an alkanediol di(meth)acrylate.
- Furthermore, in the present invention, the monomer mixture preferably contains the acyloxystyrene in a proportion of 80 to 800 μmol/g and the di(meth)acrylate compound in a proportion of 200 to 2,000 μmol/g.
- Moreover, in the present invention, preferably, the organic solvent is at least one member selected from the group consisting of a hydrocarbon and an alcohol, and the organic solvent is used in a weight ratio of 0.5 to 2.5 based on the monomer mixture.
- According to the present invention, as described above, an aromatic vinyl compound-acyloxystyrene-di(meth)acrylate compound copolymer is produced by a suspension copolymerization of a monomer mixture containing an aromatic vinyl compound, an acyloxystyrene and a di(meth)acrylate compound in predetermined proportions, respectively, and the acyloxy group of the acyloxystyrene monomer component in the copolymer is then hydrolyzed, whereby a porous resin bead containing an aromatic vinyl compound-hydroxystyrene-di(meth)acrylate copolymer is obtained.
- Accordingly, the porous resin bead obtained by the method of the present invention has appropriate crosslink density and distribution of hydroxyl groups. Therefore, when this bead is used as a support for solid phase synthesis of oligonucleotide, the reactions performed on the support are not inhibited with each other, so that oligonucleotide can be produced through a solid-phase synthesis with high purity at a high synthesis rate. In particular, according to the present invention, oligonucleotide can be obtained in a high synthesis yield while keeping the proportion of full-length oligonucleotide high.
- The method for producing a porous resin bead containing an aromatic vinyl compound-hydroxystyrene-di(meth)acrylate copolymer of the present invention includes dissolving a monomer mixture containing an aromatic vinyl compound, an acyloxystyrene and a di(meth)acrylate compound, and a polymerization initiator in an organic solvent to obtain a solution of the monomer mixture and the polymerization initiator; suspending the solution in water in the presence of a dispersion stabilizer; performing a suspension copolymerization to thereby obtain an aromatic vinyl compound-acyloxystyrene-di(meth)acrylate compound copolymer; and hydrolyzing the acyloxy group of the acyloxystyrene monomer component in the obtained copolymer.
- In the present invention, examples of the aromatic vinyl compound include, but are not limited to, styrene, a nucleus alkyl-substituted styrene such as o-methylstyrene, m-methylstyrene, p-methylstyrene, 2,4-dimethylstyrene, ethylstyrene and p-tert-butylstyrene, an α-alkyl-substituted styrene such as α-methylstyrene and α-methyl-p-methylstyrene, and a nucleus halogenated styrene such as chlorostyrene. According to the present invention, among all, styrene is preferably used as the aromatic vinyl compound.
- In the present invention, the acyloxystyrene may have, in addition to the acyloxy group, one or a plurality of arbitrary substituents on the aromatic nucleus so long as they do not adversely affect the suspension copolymerization or give no harmful effect when the obtained porous bead is used as a support for solid phase synthesis. Examples of such substituents include an alkyl group or an alkoxyl group having a carbon number of 1 to 5, a halogen atom and a nitro group.
- In the present invention, the position of the acyloxy group on the aromatic nucleus of the acyloxystyrene with respect to the vinyl group is not particularly limited but is preferably the para-position. That is, in the present invention, the acyloxystyrene is preferably a p-acyloxystyrene. Further, the alkyl group in the acyl group preferably has a carbon number of 1 to 5. Particularly, in the present invention, p-acetoxystyrene is preferably used as the acyloxystyrene.
- In the present invention, the di(meth)acrylate compound means a dimethacrylate or a diacrylate and is preferably at least one member selected from the group consisting of a (poly)ethylene glycol di(meth)acrylate and an alkanediol di(meth)acrylate.
- Specific examples of the (poly)ethylene glycol di(meth)acrylate include ethylene glycol di(meth)acrylate, diethylene glycol di(meth)acrylate, triethylene glycol di(meth)acrylate, tetraethylene glycol di(meth)acrylate, polyethylene glycol di(meth)acrylate, propylene glycol di(meth)acrylate, dipropylene glycol di(meth)acrylate, tripropylene glycol di(meth)acrylate, and polypropylene glycol di(meth)acrylate.
- In the present invention, the alkane in the alkanediol di(meth)acrylate preferably has a carbon number of 4 to 10. Accordingly, specific examples of the alkanediol di(meth)acrylate include 1,2-butanediol di(meth)acrylate, 1,3-butanediol di(meth)acrylate, 1,4-butanediol di(meth)acrylate, 1,5-pentanediol di(meth)acrylate, 1,6-hexanediol di(meth)acrylate, 1,9-nonanediol di(meth)acrylate, 1,10-decanediol di(meth)acrylate, 2-methyl-1,8-octanediol di(meth)acrylate, and 1,4-cyclohexanediol di(meth)acrylate.
- According to the present invention, in the monomer mixture of an aromatic vinyl compound, an acyloxystyrene and a di(meth)acrylate compound, which is used for the suspension copolymerization, the acyloxystyrene is preferably contained in a proportion of 80 to 800 μmol/g, and more preferably from 200 to 500 μmol/g. If the proportion of the acyloxystyrene in the monomer mixture is less than 80 μmol/g, the porous resin bead to be obtained is low in the amount of the hydroxyl group and when used as a support for solid phase synthesis, the amount of the synthesis reaction product to be obtained is small. On the other hand, if the proportion of the acyloxystyrene in the monomer mixture exceeds 800 μmol/g, since the hydroxyl group density in the porous resin bead to be obtained is high, chemical reactions occurring adjacently to each other on the support are inhibited with each other, resulting in reduction in the purity of the synthesis reaction product to be obtained.
- In the monomer mixture, the proportion of the di(meth)acrylate compound is preferably from 200 to 2,000 μmol/g, and more preferably from 300 to 1,500 μmol/g. If the proportion of the di(meth)acrylate compound in the monomer mixture is less than 200 μmol/g, the specific surface area of the porous resin bead to be obtained is extremely small and when used as a support for solid phase synthesis, the amount of the synthesis reaction product to be obtained is small. On the other hand, if the proportion of the di(meth)acrylate compound in the monomer mixture exceeds 2,000 μmol/g, since the swelling degree of the porous resin bead to be obtained in an organic solvent is low, the amount of the synthesis reaction product to be obtained is small.
- In the present invention, the suspension copolymerization of the monomer mixture containing an aromatic vinyl compound, an acyloxystyrene and a di(meth)acrylate compound can be performed by a conventionally known normal method. For example, the monomer mixture and a polymerization initiator are dissolved in an organic solvent, a dispersion stabilizer is separately dissolved in water, these two solutions are mixed in a nitrogen stream, the mixed solution is stirred so that the solution obtained by dissolving the monomer mixture and the polymerization initiator in the organic solvent can be dispersed as fine droplets in water, and the copolymerization is performed with stirring by raising the temperature. The suspension copolymerization reaction may be performed usually with stirring at a reaction temperature of 60 to 90° C. over 0.5 to 48 hours, but the copolymerization reaction conditions are not limited thereto.
- In the present invention, at least one member selected from the group consisting of a hydrocarbon and an alcohol is used as the organic solvent. In the present invention, the hydrocarbon includes an aliphatic hydrocarbon and an aromatic hydrocarbon, and the aliphatic hydrocarbon may be either saturated or unsaturated but is preferably an aliphatic saturated hydrocarbon having a carbon number of 5 to 12 or an alkylbenzene. Examples of the aliphatic saturated hydrocarbon having a carbon number of 5 to 12 include n-hexane, n-heptane, n-octane, isooctane, undecane and dodecane. Examples of the alkylbenzene include toluene.
- On the other hand, the alcohol is preferably an aliphatic alcohol, and more preferably an aliphatic alcohol having a carbon number of 5 to 12. Specific preferred examples thereof include 2-ethylhexyl alcohol, tert-amyl alcohol, nonyl alcohol, 2-octanol, decanol, lauryl alcohol and cyclohexanol. In the present invention, one of these hydrocarbons and alcohols may be used alone, or the hydrocarbon and the alcohol may be used in combination.
- In the present invention, the organic solvent is usually used in a weight ratio of organic solvent/monomer of 0.5 to 2.5, preferably from 1.0 to 2.0. If the weight ratio of organic solvent/monomer is less than or exceeds the above-mentioned range, in either case, the specific surface area of the porous resin bead to be obtained becomes small and, for example, when used as a support for solid phase synthesis, the amount of the synthesis reaction product by a chemical reaction is reduced.
- The dispersion stabilizer is not particularly limited, and an arbitrary dispersion stabilizer may be used so long as it is conventionally used as a dispersion stabilizer in the suspension polymerization. Examples of the dispersion stabilizer include a hydrophilic protective colloid agent such as polyvinyl alcohol, polyacrylic acid, gelatin, starch and carboxyl methyl cellulose; and a hardly-soluble inorganic powder such as calcium carbonate, magnesium carbonate, calcium phosphate, barium sulfate, calcium sulfate and bentonite. Although not particularly limited, the amount of the dispersion stabilizer used is preferably from 0.01 to 10 wt % based on the water used in the suspension polymerization. If the amount of the dispersion stabilizer is less than 0.01 wt % based on the water used in the suspension polymerization, the stability of suspension polymerization is impaired and a large amount of an aggregate is produced. On the other hand, if the amount of the dispersion stabilizer exceeds 10 wt % based on the water used in the suspension polymerization, a large number of particles which are too fine to use as a support for solid phase synthesis are produced.
- Further, the polymerization initiator for use in the suspension polymerization is not particularly limited, and an arbitrary polymerization initiator may be used so long as it is conventionally used as a polymerization initiator in suspension polymerization. Examples of the polymerization initiator include a peroxide such as dibenzoyl peroxide, dilauroyl peroxide, distearoyl peroxide, 1,1-di(tert-butylperoxy)-2-methylcyclohexane, 1,1-di(tert-hexylperoxy)-3,3,5-trimethylcyclohexane, 1,1-di(tert-hexylperoxy)cyclohexane, 1,1-di(tert-butylperoxy)cyclohexane, di-tert-hexyl peroxide, tert-butylcumyl peroxide, di-tert-butyl peroxide, 1,1,3,3-tetramethylbutylperoxy-2-ethylhexanoate, tert-hexylperoxy-2-ethylhexanoate, tert-butylperoxy-2-ethylhexanoate and tert-butylperoxyisopropyl monocarbonate; and an azo compound such as 2,2′-azobisisobutyronitrile, 2,2′-azobis-2-methylbutyronitrile and 2,2′-azobis-2,4-dimethylvaleronitrile.
- As described above, the organic solvent in which the monomer mixture and the polymerization initiator are dissolved is suspended in water and subjected to suspension copolymerization to precipitate a copolymer. Thereafter, the copolymer is purified by washing to remove monomers remaining in the copolymer and impurities such as organic solvent, dispersion stabilizer and polymerization initiator. The solvent for washing is not particularly limited so long as it is a solvent useful for the removal of those impurities, but water, methanol, ethanol, acetonitrile, acetone, toluene, hexane, tetrahydrofuran or the like is usually used. For washing the obtained copolymer, this may be attained, for example, by suction-filtering the liquid dispersion of the copolymer after suspension copolymerization, washing the resulting copolymer with stirring in the solvent for washing, and repeating the operation of suction-filtering and subsequent washing of the copolymer in a similar manner. In the process of this washing, the system may be heated, if necessary, to remove volatile impurities in the copolymer.
- According to the present invention, an aromatic vinyl compound-acyloxystyrene-di(meth)acrylate compound copolymer is firstly obtained in this way, and the acyloxy group of the acyloxystyrene monomer component in the copolymer is then hydrolyzed, whereby the objective porous resin bead containing an aromatic vinyl compound-hydroxystyrene-di(meth)acrylate copolymer can be obtained.
- For hydrolyzing the acyloxy group of the acyloxystyrene monomer component in the aromatic vinyl compound-acyloxystyrene-di(meth)acrylate compound copolymer, conventionally known methods and conditions may be employed. However, in the present invention, it is not necessary to hydrolyze the acyloxy groups of all acyloxystyrene monomer components in the obtained aromatic vinyl compound-acyloxystyrene-di(meth)acrylate compound copolymer, and it may be usually sufficient if a part of the acyloxy groups are hydrolyzed.
- In the present invention, the catalyst used for the hydrolysis of the acyloxy group of the acyloxystyrene monomer component in the aromatic vinyl compound-acyloxystyrene-di(meth)acrylate compound copolymer may be an acid catalyst such as hydrochloric acid and hydrobromic acid, or an alkali catalyst such as hydrazine, sodium hydroxide, tetramethylammonium hydroxide and aqueous ammonia. Preferred examples of the reaction solvent used for the hydrolysis include, but are not limited to, tetrahydrofuran, ethanol, methanol, dioxane and water.
- In the present invention, the reaction conditions of the hydrolysis may be appropriately set. For example, in the case of performing the hydrolysis by using an aqueous sodium hydroxide solution containing ethanol, the amount of sodium hydroxide used is from 1 to 10 equivalents of the acyloxystyrene monomer component in the aromatic vinyl compound-acyloxystyrene-di(meth)acrylate compound copolymer, the amount of ethanol used is from 1 to 10 times the weight of water used, and the reaction is performed at 50 to 80° C. for 1 to 12 hours. Preferably, the amount of sodium hydroxide used is from 1 to 5 equivalents of the acyloxystyrene monomer component, and the reaction is performed at 50 to 80° C. for 1 to 6 hours. If the amount of sodium hydroxide used is too large or the reaction time is too long, the ester derived from the di(meth)acrylate compound is hydrolyzed and the solvent resistance of the porous resin bead is impaired. The reaction mixture after hydrolysis may be adjusted to near neutrality by using an acid or an alkali and then washed by the same method as described above to remove impurities.
- As described above, according to the present invention, an aromatic vinyl compound-acyloxystyrene-di(meth)acrylate compound copolymer can be obtained through a suspension copolymerization of a monomer mixture containing an aromatic vinyl compound, an acyloxystyrene and a di(meth)acrylate compound, and a porous resin bead containing an aromatic vinyl compound-hydroxystyrene-di(meth)acrylate compound copolymer can be produced by hydrolyzing the acyloxy group of the acyloxystyrene monomer component in the obtained copolymer. Thereafter, if desired, the porous resin bead may be further subjected to a treatment such as drying or classification.
- The thus-obtained porous resin bead can be suitably used as a support for solid phase synthesis to synthesize, for example, oligonucleotide. Incidentally, the oligonucleotide synthesis using the porous resin bead according to the present invention as a support for solid phase synthesis can be performed by a conventionally known method. For example, a linker is bound to the hydroxy group of the porous resin bead of the present invention, and amidite units are then bound one by one to give a predetermined base sequence from the end of the linker. This synthesis reaction can be performed using an automatic synthesis apparatus. For example, a linker-bound porous resin particle is packed in a flow-type reactor of the apparatus, various organic solvents such as acetonitrile and an amidite solution are sequentially fed thereto, and the reaction is repeated. Finally, the linker portion is cut off by hydrolysis or the like, whereby the objective oligonucleotide can be obtained. As for the linker, a conventionally known linker is appropriately used. For example, the oligonucleotide can be synthesized by binding a nucleoside linker having a structure shown below to the porous resin particle of the present invention.
- Herein, in the above-described structural formula, the circle represents the porous resin bead containing an aromatic vinyl compound-hydroxystyrene-di(meth)acrylate copolymer, which is obtained by the method of the present invention, DMT represents a protective dimethoxytrityl group at the 5′-position, and B1 represents a base.
- The present invention is described below by referring to Examples, but the present invention is not limited to these Examples.
- A 500 mL-volume separable flask equipped with a condenser, a stirrer and a nitrogen inlet tube was placed in a constant temperature water bath, and 2.5 g of polyvinyl alcohol and 250 g of distilled water were charged thereto and dissolved with stirring at 300 rpm to prepare an aqueous polyvinyl alcohol solution.
- Separately, 50 g of styrene, 3.5 g (proportion in the monomer mixture: 369 μmol/g) of p-acetoxystyrene, 5 g (proportion in the monomer mixture: 431 μmol/g) of ethylene glycol dimethacrylate, 85 g of 2-ethylhexanol, 25 g of isooctane and 1 g of benzoyl peroxide (25% aqueous product) were mixed to prepare a solution. This solution was added to the aqueous polyvinyl alcohol solution prepared above and after stirring at a stirring rate of 500 rpm in a nitrogen stream at room temperature, suspended in water, and suspension copolymerization was performed for 8 hours by raising the temperature to 80° C.
- (Washing)
- The obtained copolymer was washed by filtration using distilled water and acetone and then dispersed in acetone to make a total amount of about 1 L.
- (Classification)
- The resulting liquid dispersion was then allowed to stand still until the bead-like copolymer was precipitated so that the precipitate was not disturbed even when the liquid was tilted, and the supernatant acetone was then discarded. The operation of again adding acetone to the precipitate to make a total amount of about 500 mL, allowing the liquid dispersion to stand still, and then discarding acetone was repeated 10 times or more, thereby performing the classification. The thus-obtained liquid dispersion was filtered, and the residue was dried under reduced pressure to obtain a porous resin bead composed of a styrene-p-acetoxystyrene-ethylene glycol dimethacrylate copolymer, as powder.
- (Hydrolysis)
- A 500 mL-volume separable flask equipped with a condenser, a stirrer and a nitrogen inlet tube was dipped in a constant temperature water bath, and 20 g of the porous resin bead composed of a styrene-p-acetoxystyrene-ethylene glycol dimethacrylate copolymer, obtained above as powder, and 100 g of ethanol were charged thereto and dispersed with stirring. Subsequently, an aqueous solution prepared by dissolving 1 g of sodium hydroxide in 50 g of distilled water was added, and hydrolysis reaction was then performed for 3 hours by raising the temperature to 75° C. After the completion of reaction, the obtained reaction mixture was neutralized with hydrochloric acid and washed by filtration using distilled water and acetone. Finally, the obtained liquid dispersion was filtered and further dried under reduced pressure to obtain a porous resin bead composed of a styrene-p-hydroxystyrene-ethylene glycol dimethacrylate copolymer, as powder.
- (Analysis)
- The obtained porous resin bead was subjected to the following analyses.
- (1) Median Diameter
- The median diameter was measured by a laser diffraction/scattering method (a laser diffraction/scattering particle size distribution analyzer, LA-920, manufactured by Horiba Ltd.).
- (2) Average Pore Diameter
- The average pore diameter was measured by a mercury penetration method (a mercury porosimeter, PoreMaster 60GT, manufactured by Quantachrome Instruments).
- (3) Amount of Hydroxy Group
- The amount of hydroxy group was measured by titration according to JIS K 0070. That is, the hydroxy group of the porous resin bead to be measured was acetylated with a known amount of an acetylation reagent (acetic anhydride/pyridine), and the amount of the acetic anhydride not consumed in the actylation was determined by titration using potassium hydroxide, whereby the amount of the hydroxy group in the sample was calculated. More specifically, this was performed as follows.
- The acetylation reagent was prepared by adding pyridine to 25 g of acetic anhydride to make a total amount of 100 mL, from 0.5 to 2 g of the measurement sample (dried porous resin bead) was weighed in a flask, and 0.5 mL of the acetylation reagent and 4.5 mL of pyridine were exactly added to the flask. The mixture in the flask was kept at 95 to 100° C. for 2 hours and then allowed to cool to room temperature, and 1 mL of distilled water was added thereto. After heating for 10 minutes, the acetic anhydride not consumed in the acetylation was decomposed. The entire content in the flask was transferred to a beaker, diluted with distilled water to a total amount of 150 mL and then titrated with an aqueous 0.5 N sodium hydroxide solution.
- Separately, a blank was measured by the same operation as above without charging the measurement sample. The amount of the hydroxy group in the measurement sample was calculated according to the following formula (I), wherein A (μmol/g) is the amount of the hydroxyl group in the measurement sample, B (mL) is the titer of the aqueous potassium hydroxide solution when measuring a blank, C (mL) is the titer of the aqueous potassium hydroxide solution when measuring the measurement sample, f is the factor of the aqueous potassium hydroxide solution, and M (g) is the weight of the measurement sample weighed.
-
A=(B−C)×0.5(mol/L)×f×1000÷M (1) - The median diameter of the porous resin bead was 80 μm, the average pore diameter was 29 nm, and the amount of the hydroxy group was 491 μmol/g.
- A porous resin bead composed of a styrene-p-hydroxystyrene-triethylene glycol dimethacrylate copolymer was obtained as powder in the same manner as in Example 1 except that in Example 1, 40 g of styrene, 3.5 g (proportion in the monomer mixture: 369 μmol/g) of p-acetoxystyrene, 15 g (proportion in the monomer mixture: 897 μmol/g) of triethylene glycol dimethacrylate, 80 g of 1-undecanol and 1 g of benzoyl peroxide (25% aqueous product) were used.
- The obtained porous resin bead was analyzed in the same manner as in Example 1, and as a result, the median diameter was 75 μm, the average pore diameter was 34 nm, and the amount of the hydroxy group was 393 μmol/g.
- (Binding of Nucleoside Linker to Porous Resin Bead)
- The porous resin bead (1 g) obtained above, 0.18 g of DMT-dT-3′-succinate (produced by Beijing OM Chemicals), 0.09 g of HBTU (produced by Novabiochem), 0.084 mL of N,N-diisopropylethylamine and 10 mL of acetonitrile were mixed and reacted with stirring at room temperature for 12 hours, and the reaction mixture was washed by filtering using acetonitrile and dried. Thereto, 2.5 mL of CapA (20% acetic anhydride/80% acetonitrile), 2.5 mL of CapB (20% N-methylimidazole/30% pyridine/50% acetonitrile), 0.025 g of 4-dimethylaminopyridine and 5 mL of acetonitrile were mixed and reacted with stirring at room temperature for 12 hours, and the reaction mixture was washed by filtering using acetonitrile and then dried under reduced pressure to obtain a DMT-dT-3′-succinate-bound porous resin bead. The addition amount of the DMT-dT-3′-succinate determined by measuring the absorbance (412 nm) of the DMT group deprotected using a p-toluenesulfonic acid/acetonitrile solution is shown in Table 1.
- (Synthesis of Oligonucleotide)
- The DMT-dT-3′-succinate-bound porous resin bead obtained above was packed in a column, the column was set in an Applied Biosystems 3400 DNA synthesizer (manufactured by Applied Biosystems), and synthesis of oligonucleotide dT20 was performed under the conditions of a synthesis scale of 1 μmol and DMT-off. The column after the synthesis was dried, and the weight yield (mg/μmol) of the obtained oligonucleotide determined by the weight measurement is shown in Table 1.
- Excision and deprotection of oligonucleotide from the porous resin bead were performed by a reaction using concentrated aqueous ammonia at 55° C. for 12 hours. The OD yield of the obtained oligonucleotide as determined by ultraviolet absorbance measurement (260 nm) is shown in Table 1.
- The proportion of dT20 (full-length) in the obtained oligonucleotide as determined by HPLC measurement (Alliance UV System manufactured by Waters Corp., Hydrosphere C18 manufactured by YMC) is shown in Table 1.
- A porous resin bead composed of a styrene-p-hydroxystyrene-1,6-hexanediol dimethacrylate copolymer was obtained as powder in the same manner as in Example 1 except that in Example 1, 40 g of styrene, 3.5 g (proportion in the monomer mixture: 369 μmol/g) of p-acetoxystyrene, 15 g (proportion in the monomer mixture: 1,008 μmol/g) of 1,6-hexanediol dimethacrylate, 75 g of 1-decanol and 1 g of benzoyl peroxide (25% aqueous product) were used.
- The obtained porous resin bead was analyzed in the same manner as in Example 1, and as a result, the median diameter was 72 μm, the average pore diameter was 26 nm, and the amount of the hydroxy group was 446 μmol/g.
- A porous resin bead composed of a styrene-p-hydroxystyrene-1,9-nonanediol diacrylate copolymer was obtained as powder in the same manner as in Example 1 except that in Example 1, 48 g of styrene, 2.5 g (proportion in the monomer mixture: 264 μmol/g) of p-acetoxystyrene, 8 g (proportion in the monomer mixture: 510 μmol/g) of 1,9-nonanediol diacrylate, 90 g of 1-nonanol and 1 g of benzoyl peroxide (25% aqueous product) were used.
- (Binding of Nucleoside Linker to Porous Resin Bead and Synthesis of Oligonucleotide)
- Production of a DMT-dT-3′-succinate-bound porous resin bead and synthesis of oligonucleotide dT20 were performed in the same manner as in Example 2. The bound amount of DMT-dT-3′-succinate, the weight yield of oligonucleotide, the OD amount of oligonucleotide, and the proportion of dT20 (full-length) are shown in Table 1.
- A porous resin bead composed of a styrene-p-hydroxystyrene-divinylbenzene copolymer was obtained as powder in the same manner as in Example 1 except that in Example 1, 48 g of styrene, 3.5 g (proportion in the monomer mixture: 369 μmol/g) of p-acetoxystyrene, 7 g (proportion in the monomer mixture: 506 μmol/g) of divinylbenzene (55%), 52 g of 2-ethylhexanol, 23 g of isooctane and 1 g of benzoyl peroxide (25% aqueous product) were used.
- The obtained porous resin bead was analyzed in the same manner as in Example 1, and as a result, the median diameter was 86 μm, the average pore diameter was 33 nm, and the amount of the hydroxy group was 422 μmol/g.
- (Binding of Nucleoside Linker to Porous Resin Bead and Synthesis of Oligonucleotide)
- Production of a DMT-dT-3′-succinate-bound porous resin bead and synthesis of oligonucleotide dT20 were performed in the same manner as in Example 2. The bound amount of DMT-dT-3′-succinate, the weight yield of oligonucleotide, the OD amount of oligonucleotide, and the proportion of dT20 (full-length) are shown in Table 1.
- A porous resin bead composed of a styrene-p-hydroxystyrene-divinylbenzene copolymer was obtained as powder in the same manner as in Example 1 except that in Example 1, 48 g of styrene, 3.5 g (proportion in the monomer mixture: 369 μmol/g) of p-acetoxystyrene, 7 g (proportion in the monomer mixture: 506 μmol/g) of divinylbenzene (55%), 75 g of 1-decanol and 1 g of benzoyl peroxide (25% aqueous product) were used.
- The obtained porous resin bead was analyzed in the same manner as in Example 1, and as a result, the median diameter was 116 μm, the average pore diameter was 68 nm, and the amount of the hydroxy group was 432 μmol/g.
- (Binding of Nucleoside Linker to Porous Resin Bead and Synthesis of Oligonucleotide)
- Production of a DMT-dT-3′-succinate-bound porous resin bead and synthesis of oligonucleotide dT20 were performed in the same manner as in Example 2. The bound amount of DMT-dT-3′-succinate, the weight yield of oligonucleotide, the OD amount of oligonucleotide, and the proportion of dT20 (full-length) are shown in Table 1.
-
TABLE 1 Compara- Compara- Exam- Exam- tive tive ple ple Example Example 2 4 1 2 Median diameter (μm) 75 83 86 116 Average pore diameter (nm) 34 75 33 68 Amount of hydroxy group 393 350 422 432 (μmol/g) Bound amount of DMT-dT- 207 184 200 198 3′-succinate (μmol/g) Weight yield of 6.7 6.6 5.7 5.4 oligonucleotide (μmol/g) OD Yield of oligonucleotide 197 191 162 150 (OD/μmol) Proportion of dT20 (full- 95 94 93 92 length) (%) - As apparent from the results in Table 1, when the porous resin beads of Examples 2 to 4 according to the present invention are used, as compared with the porous resin beads of Comparative Examples 1 and 2, the synthesis yield of oligonucleotide can be increased while maintaining a high full-length percentage.
- While the present invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the scope thereof.
- This application is based on Japanese patent application No. 2007-286835 filed on Nov. 5, 2007, the entire contents thereof being hereby incorporated by reference.
- Further, all references cited herein are incorporated in their entireties.
Claims (4)
1. A method for producing a porous resin bead containing an aromatic vinyl compound-hydroxystyrene-di(meth)acrylate copolymer, the method comprising:
dissolving a monomer mixture containing an aromatic vinyl compound, an acyloxystyrene and a di(meth)acrylate compound, and a polymerization initiator in an organic solvent to obtain a solution containing the monomer mixture and the polymerization initiator;
suspending the solution in water in the presence of a dispersion stabilizer;
performing a suspension copolymerization to thereby obtain an aromatic vinyl compound-acyloxystyrene-di(meth)acrylate compound copolymer; and
hydrolyzing an acyloxy group of the acyloxystyrene monomer component in said copolymer.
2. The method for producing a porous resin bead according to claim 1 , wherein the di(meth)acrylate compound is at least one member selected from the group consisting of a (poly)ethylene glycol di(meth)acrylate and an alkanediol di(meth)acrylate.
3. The method for producing a porous resin bead according to claim 1 , wherein the monomer mixture contains the acyloxystyrene in a proportion of 80 to 800 μmol/g and the di(meth)acrylate compound in a proportion of 200 to 2,000 μmol/g.
4. The method for producing a porous resin bead according to claim 1 , wherein the organic solvent is at least one member selected from the group consisting of a hydrocarbon and an alcohol, and the organic solvent is used in a weight ratio of 0.5 to 2.5 based on the monomer mixture.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13/235,869 US20120010396A1 (en) | 2007-11-05 | 2011-09-19 | Production method of porous resin bead |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2007-286835 | 2007-11-05 | ||
JP2007286835A JP5210597B2 (en) | 2007-11-05 | 2007-11-05 | Method for producing porous resin beads |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
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US13/235,869 Continuation US20120010396A1 (en) | 2007-11-05 | 2011-09-19 | Production method of porous resin bead |
Publications (1)
Publication Number | Publication Date |
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US20090118383A1 true US20090118383A1 (en) | 2009-05-07 |
Family
ID=40292480
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/264,940 Abandoned US20090118383A1 (en) | 2007-11-05 | 2008-11-05 | Production method of porous resin bead |
US13/235,869 Abandoned US20120010396A1 (en) | 2007-11-05 | 2011-09-19 | Production method of porous resin bead |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
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US13/235,869 Abandoned US20120010396A1 (en) | 2007-11-05 | 2011-09-19 | Production method of porous resin bead |
Country Status (6)
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US (2) | US20090118383A1 (en) |
EP (1) | EP2055724B1 (en) |
JP (1) | JP5210597B2 (en) |
CN (1) | CN101429261B (en) |
AT (1) | ATE482237T1 (en) |
DE (1) | DE602008002678D1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090291294A1 (en) * | 2008-05-26 | 2009-11-26 | Nitto Denko Corporation | Solid-phase support for nucleic acid synthesis |
US20140096328A1 (en) * | 2011-06-09 | 2014-04-10 | Haier Group Corporation | Solid particle for washing and washing method using the same |
US10426712B2 (en) * | 2014-09-17 | 2019-10-01 | Kuraray Noritake Dental Inc. | Dental polymerizable composition |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2666794A1 (en) * | 2012-05-23 | 2013-11-27 | Nitto Denko Corporation | Solid-phase support for oligonucleotide synthesis and oligonucleotide synthesis method |
CN104693333B (en) * | 2013-12-04 | 2019-05-14 | 日东电工株式会社 | Porous resin bead and the method that nucleic acid is prepared by using it |
CZ307994B6 (en) * | 2014-06-09 | 2019-10-09 | Ústav Chemických Procesů Av Čr, V. V. I. | Styrenic polymer and preparing it |
EP3315516A1 (en) | 2016-10-31 | 2018-05-02 | University of Vienna | Macroporous beads |
CN108250372B (en) * | 2018-01-12 | 2020-09-11 | 南京大学 | Hydrophilic magnetic styrene strong base anion exchange microsphere resin and preparation method thereof |
WO2023069599A1 (en) * | 2021-10-22 | 2023-04-27 | Hongene Biotech Corporation | Polymeric solid support for oligonucleotide synthesis |
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US5461017A (en) * | 1991-12-13 | 1995-10-24 | Mobil Oil Corporation | Olefin polymerization catalysts |
US20050256285A1 (en) * | 2004-05-14 | 2005-11-17 | Nitto Denko Corporation | Production method of porous resin |
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JPS5223193A (en) | 1975-08-15 | 1977-02-21 | Cosmo Co Ltd | Preparation of microporous resin |
JPS58210914A (en) | 1982-06-02 | 1983-12-08 | Cosmo Co Ltd | Preparation of macroporous resin |
JPH0586132A (en) * | 1991-09-30 | 1993-04-06 | Mitsubishi Kasei Corp | Porous resin and its production |
EP0496405A1 (en) * | 1991-01-25 | 1992-07-29 | Mitsubishi Kasei Corporation | Porous resin and process for its production |
JP2987949B2 (en) * | 1991-01-25 | 1999-12-06 | 三菱化学株式会社 | Porous resin and method for producing the same |
JP4602011B2 (en) * | 2003-08-29 | 2010-12-22 | 日東電工株式会社 | Porous resin beads and method for producing the same |
JP4890457B2 (en) * | 2004-09-02 | 2012-03-07 | アイシス ファーマシューティカルズ, インコーポレーテッド | Polymer beads for oligomer synthesis |
CN1297579C (en) * | 2005-02-28 | 2007-01-31 | 天津南开和成科技有限公司 | Crosslinked polystyrene gel and use thereof |
JP2007286835A (en) | 2006-04-14 | 2007-11-01 | Victor Co Of Japan Ltd | Voice content distribution system and content reception/reproduction terminal |
-
2007
- 2007-11-05 JP JP2007286835A patent/JP5210597B2/en active Active
-
2008
- 2008-11-04 EP EP20080019288 patent/EP2055724B1/en active Active
- 2008-11-04 AT AT08019288T patent/ATE482237T1/en not_active IP Right Cessation
- 2008-11-04 DE DE200860002678 patent/DE602008002678D1/en active Active
- 2008-11-05 US US12/264,940 patent/US20090118383A1/en not_active Abandoned
- 2008-11-05 CN CN2008101744314A patent/CN101429261B/en active Active
-
2011
- 2011-09-19 US US13/235,869 patent/US20120010396A1/en not_active Abandoned
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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US5461017A (en) * | 1991-12-13 | 1995-10-24 | Mobil Oil Corporation | Olefin polymerization catalysts |
US20050256285A1 (en) * | 2004-05-14 | 2005-11-17 | Nitto Denko Corporation | Production method of porous resin |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090291294A1 (en) * | 2008-05-26 | 2009-11-26 | Nitto Denko Corporation | Solid-phase support for nucleic acid synthesis |
US20140096328A1 (en) * | 2011-06-09 | 2014-04-10 | Haier Group Corporation | Solid particle for washing and washing method using the same |
US9315766B2 (en) * | 2011-06-09 | 2016-04-19 | Haier Group Corporation | Solid particle for washing and washing method using the same |
US10426712B2 (en) * | 2014-09-17 | 2019-10-01 | Kuraray Noritake Dental Inc. | Dental polymerizable composition |
Also Published As
Publication number | Publication date |
---|---|
JP2009114270A (en) | 2009-05-28 |
EP2055724B1 (en) | 2010-09-22 |
DE602008002678D1 (en) | 2010-11-04 |
ATE482237T1 (en) | 2010-10-15 |
CN101429261A (en) | 2009-05-13 |
CN101429261B (en) | 2011-12-07 |
JP5210597B2 (en) | 2013-06-12 |
EP2055724A1 (en) | 2009-05-06 |
US20120010396A1 (en) | 2012-01-12 |
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